Polymer affinity matrix, a method for the production and use thereof

ABSTRACT

A polymer affinity matrix for the binding of one or more substances in a fluid for removing said substance(s) from the fluid and/or decreasing the amount or concentration thereof in said fluid with a view to preventing, eliminating, or reducing undesired activation of components in said fluid is described, as well as a method for removing said substance(s) from the fluid and/or decreasing the amount or concentration thereof in said fluid, a method for the production of said matrix, use of said matrix and a kit comprising said matrix. The polymer affinity matrix comprises a solid support, a space and a ligand, containing arginine as a binding unit.

TECHNICAL FIELD

The present invention relates to a polymer affinity matrix for bindingone or more substances in a fluid for removing said substance(s) fromthe fluid and/or decreasing the amount or concentration thereof in saidfluid with a view to preventing, eliminating, or reducing undesiredactivation of components or processes in said fluid, to a method forremoving said substance(s) from the fluid and/or decreasing the amountor concentration thereof in said fluid, to a method for producing saidmatrix, to use of said matrix and to a kit comprising said matrix.

The present invention also relates to use a polymer matrix forproduction of a polymer affinity matrix for removal of one ore moresubstances from a fluid or decreasing the amount or concentrationthereof in said fluid.

BACKGROUND OF THE INVENTION

Extracorporeal Treatment

Extracorporeal treatment of a fluid, such as blood or any other bodyfluid, requires that the fluid is brought into contact with material inthe form of e.g. tubes, lines, beads or membranes. Introduction of suchmaterial implies the use of foreign and thus potentially bioincompatible(e.g. immunologically active or procoagulatory) material. This use offoreign material is associated with activation of the host immunesystem, e.g. of components in the blood such as lymphocytes, plateletsor different types of plasma protein cascades such as proteins in thecomplement or coagulation cascade. Also, damage to cells such asmechanical or stress-induced damage to e.g. erythrocytes might causehaemolysis and subsequent life-threatening complications to the patient.Treatment of a fluid, such as blood or any body fluid, thereforerequires the use of highly biocompatible material to avoid undesiredactivation of components in said fluid, e.g. blood.

Bacterial Toxins

Bacterial endo- and exotoxins promote an overwhelming inflammatoryimmune reaction in a host due to activation caused by multipleinteractions between blood cells and soluble proteins in the host andsaid endotoxin. This immune response is described as an essentialfeature in the clinical symptoms of SIRS (systemic inflammatory responsesyndrome), sepsis or septic shock. The pathogenesis is severe and thecondition leads to tissue damage, multiple organ failure, and deathinduced by sepsis. Searching for new therapeutical drugs and methods forthe treatment of septic patients is important, since studies of recenttherapeutical interventions (Dinarello et al. in European CytokineNetwork 1997; 8:294) show a failure to protect patients with severesepsis or septic shock. Also, when manufacturing therapeuticalsubstances or fluids, care must be taken that the product isnon-pyrogenic, i.e. that the endotoxin concentration is absent or belowaccepted limits for excerting its effects.

Endotoxins

Endotoxins or “pyrogens” from the outer layers of the cell membrane ofGram-negative bacteria, e.g. Esherichia coli, Salmonella typhi,Pseudomonas aeruginosa or Proteus vulgaris, play an important role inthe pathogenesis of sepsis, septic shock and systemic inflammatoryresponse syndrome (SIRS). Endotoxins, e.g. lipopolysaccharide (LPS) fromE. coli, are composed of a lipid A and a polysaccharide chain (Zähringeret al. Adv. Carb. Chem. Biochem., 1994). The molecular structure of LPSis shown in FIG. 1. The lipid A component is the biologically mostactive part and mediates the toxic effect of endotoxins on cells. LipidA is highly conserved within the different strains of Gram-negativebacteria whereas the polysaccharide part is much more variable.

Lipid A from E. coli consists of two glucoseamine moieties, whichcontain two negatively charged phosphate groups at opposite ends of thetwo glucoseamines, and six long-chain highly hydrophobic fatty acidresidues which are linked to the two glucoseamine rings. The variablepolysaccharide chain of the endotoxin is also linked to one of theglucoseamines.

Removal of Endotoxins

The removal of endotoxins is difficult and often includes problems withrecovery of or damage to valuable proteins, i.e. proteins not intendedto be removed, or biocompatibility problems with the blood or body fluidand the means used for removal of the endotoxin. Such a biocompatibilityproblem is caused by a mere activation of the defence mechanisms of thehost and involves multiple cellular activation and release of solubleproteins such as cytokines and proteins in the complement cascade. Thecellular activation and protein release may lead to severe inflammation,i.e. systemic sepsis or septic shock, with tissue damage and organfailure as a result. Blood clotting caused by coagulation is anotherproblem caused by bioincompatibility. Moreover, the pyrogen might not becompletely removed.

Known methods for removing endotoxins include inactivation by the use ofheat, acid or alkali (U.S. Pat. Nos. 3,644,175, 3,659,027, and4,070,289). Such methods often compromise the quality of the finalproduct, i.e. the inactivated detoxified fluid, since the fluid and itsvaluable proteins might be denatured or inactivated as well using thistype of processes. Other methods to be used include adsorption tocharcoal, or oxidative decomposition using an oxidising agent, e.g.potassium permanganate, aqueous hydrogen peroxide, and sodiumhypochlorite.

Conventional Means for Removal of Endotoxins

Extracorporeal removal of endotoxins' from plasma or whole blood ofseptic patients is discussed as a potential therapeutic strategy in thetreatment of sepsis, septic shock and SIRS. In human plasma there areseveral binding proteins that play a role in the mediation andinhibition of endotoxin effects on cells, e.g. lipopolysaccharidebinding protein (LBP), bactericidal permeability increasing protein(BPI), sCD14, CAP18, and lactoferrin. The peptide antibiotic polymyxin B(produced by Bacillus polymyxa) inhibits the action of endotoxins oncells. The horse shoe crab (Limulus polyphemus) also containsendotoxin-binding proteins and a cell lysate from this species is usedfor the detection of endotoxins (Limulus amebocyte lysate, LAL, orLimulus test). The binding motifs of such endotoxin binding proteins maybe used for extracorporeal treatment of plasma or whole blood of septicpatients.

A common characteristic of many endotoxin binding sequences is thepresence of alternating positively charged and hydrophobic amino acidsin the binding part thereof. It has been shown for one endotoxin bindingprotein originating from the horse shoe crab that the endotoxin bindingsequence forms an amphipatic secondary structure where positivelycharged residues and hydrophobic residues are located at opposite facesof a loop structure (Hoess et al. EMBO J, 1993). A similar pattern hasbeen proposed for other endotoxin binding sites.

Positively charged polymers such as polyethyleneimine (Mizner et al.Artif. Organs, 1993; Weber et al. ASAIO J, 1995; Petsch et al. JChromatogr. Biomed. Sci. Appl., 1998) or diethylaminoethyl (DEAE)modified cellulose (Weber et al. A.S.A.I.O. J., 1995) have a certainadsorption capacity for endotoxins, probably based on the interaction ofthe positive charges with the negatively charged phosphate residues oflipid A. A drawback of polyethyleneimine is the high absorption ofheparin and its well-described interaction with platelets which givesrise to bioincompatibility problems such as coagulation in an in or exvivo application.

Similarly, positively charged membrane filters are used for purificationof infusion fluids. Absorption is thus dependent on attractive chargesand is less specific and may therefore remove desired valuable proteinsas well.

Arginine immobilised on sepharose has been suggested for removal ofendotoxins from plasma, blood and pharmaceutical solutions (EP 0 494 848and EP 0 333 474).

WO 92/11847 describes the use of a compound for the preparation of amedicament to be used orally, intravenously, intramuscularly,intracutaneously or intraperitoneally for the treatment of endotoxininduced effects as well as a method for the treatment of endotoxininduced effects. The compound described in WO 92/11847 is notimmobilised on a solid support.

Also, hydrophobic polymers (e.g. polystyrene, polyamide, polysulfone,and polyethersulfone) can adsorb endotoxins in aqueous solutions. Thisproperty is used in ultrafiltration membranes for purification of waterand dialysis/infusion fluid, i.e. solutions with low a or no proteincontent (Weber et al., Int. J. Artific. Org., 1997). However, theremoval of endotoxins from blood or plasma introduces a competingproblem with circulating plasma proteins (LBP, BPI, sCD14) and ofcellular receptors (e.g. CD14) to an adsorber matrix due to the plainhydrophobic absorption which has a low specificity.

The use of positively charged or hydrophobic polymers for the removal ofendotoxins shows that the specificity of the binding and thus also theselectivity for the removal of the endotoxin in plasma is low. Inpractice it has been a challenge to develop an efficient endotoxinadsorbent with both high specificity and high selectivity, still incombination with high biocompatibility.

The use of peptide sequences from endotoxin binding proteins has beensuggested for therapeutical applications (U.S. Pat. Nos. 5,639,727 and5,643,875) by use of e.g. bactericidal/permeability-increasing (BPI)protein products. A drawback of immobilised peptides is the costs ofsynthesis and the necessity of the peptides to be immobilised withoutinterfering with the binding structure.

Affinity ligands such as histamine, histidine and polymyxin B areeffective for the removal of endotoxins though their effectiveness isdependent upon other proteins in the fluid and decreases drastically inthe presence of serum proteins like serum albumin or other negativelycharged proteins (Anspach and Hilbeck, J. Chromatogr., 1995, Petsch etal. J. Chromatogr., 1997, EP 0 129 786). However, Polymyxin B is toxicto the central nervous system and may cause kidney damage, which is adrawback in marketing approval due to a risk of leakage into the bloodof a patient. U.S. Pat. No. 4,771,104 describes an endotoxin detoxifyingmaterial comprising a fibrous carrier to which polymyxin B is fixed.

Removal of endotoxins, particularly LPS, from drugs and fluids by use ofwater-insoluble poly(ε-lysine)(PL) particles was described by Hirayamaet al, in Journal of Chromatography B, 271 (1999), 187-195.

Limitations and Future Perspectives

Efficient endotoxin removal from a protein fluid is dependent on the netcharge of the desired valuable protein from which the endotoxin shouldbe removed. The interaction between the endotoxin and its ligand is ofboth hydrophobic and ionic character, though contribution of each ofthem depends on the ionic strength and pH of the fluid. Efficientendotoxin removal is also dependent on a high perfusion andbiocompatibility of the means for removal of said endotoxin to prevente.g. cell activation or blood clotting.

However, although many means have been developed or suggested forremoval of substances such as endotoxins from fluids, e.g. blood, otherbody fluids or therapeutical fluids, none of the described means ishighly biocompatible nor has a high degree of specificity andselectivity against substances to be removed e.g. endotoxins, and at thesame time can be easily perfused, e.g. by whole blood or plasma. It isthus desirable to develop highly efficient, biocompatible, and effectivemethodologies and means for the removal of substances, e.g. endotoxins,from a fluid, and thus making it possible to avoid the problemsassociated with prior art devices. In this respect, the presentinvention addresses this need and interest.

The need for a novel and efficient method and means for decreasing theconcentrations of or removing one or more substances from a fluid, e.g.endotoxins from blood, any other body fluid or therapeutic fluid with aview to preventing, eliminating or reducing undesired activation ofcomponents or processes in a fluid is evident from the reasons describedabove, particularly within the biomedical area. Such a method and meanswould also be specifically valuable in the treatment of sepsis, septicshock and SIRS by extracorporeal removal of pyrogens or other activatingsubstances from plasma of septic patients.

Further, it is well known to bind a biospecific ligand e.g. an antibodyor a peptide to a matrix or substrate and remove by this e.g.pathophysiologically critical substances from the body or bloodcirculation.

Used known materials are e.g. sepharose (sephadex, pharmacia),polyacrylate or epoxid resins in form of beads or particles.

Known beads are for example made of polymethyl-methacrylate e.g.DALI-System (Fresenius) for LDL adsorption; Sepharose: e.g.Rheosorb-System by Plasmaselect for fibrinogen adsorption which uses animmoblized peptide; Therasorb-Immunoadsorption-System to removeautoantibodies using an immobilized sheep-antibody which binds humanimmunoglobuliris; Excorim-Protein A column (and similar systems by othermanufacturers) which uses an immobilized bacterial protein to bindimmunoglobulins.

Known membranes are for example polyamide (MAT/Merck) and others usuallydescribed as affinity membranes.

Known fabrics are for example Toray polystyrene/polyethylene meshes withPolymyxin B ligand for LPS binding.

All these substrates are modified in case of use of biological ligands(such as peptides or antibodies.) by conventional methods of wetchemistry, i.e. first the corresponding specific ligand is formed orisolated (e.g. by peptide synthesis, by biotechnological methods), thenit is purified and afterwards it is immobilized to a solid matrix.

These matrices usually do not allow solid phase synthesis of a ligand(e.g. a peptide) directly on the matrix, due to chemical incompatibilityagainst the chemicals (solvents, acids and bases used for cleavage ofprotecting groups etc) used in solid phase synthesis.

The matrices that are suitable for solid phase synthesis (e.g.polystyrene) are not suitable for therapeutic purposes due to lack ofbiocompatibility.

Therefore, a solid phase matrix, which allows solid phase synthesis aswell as contact with blood components (e.g. plasma or whole blood),would be advantageous for the following reasons:

(1) The development steps to find a suitable ligand (e.g. an optimisedpeptide structure) can be performed on the same solid matrix which canlater be used for therapeutic application. This means that the necessarybiological test procedures (e.g. measurement of affinity, specificityand binding capacity) can be performed under conditions very similar tothe conditions of the later therapeutic application. By this theinformation coming from the biological test procedures is very reliableand predictive for the later therapeutic or clinical application. Thissaves time and money and lowers risk of failure or side effects.

(2) A (peptide) ligand can be built-up by solid phase synthesis in anexactly defined way (with respect to sequence and geometry). This meansalso that the linkage (i.e. the location of the covalent connection) ofthe (peptide) ligand to the solid matrix is exactly defined.

There are somehow contradicting requirements to such a matrix withrespect to the swelling or wetting behaviour: for the solid phasesynthesis usually non-aqueous organic (polar, such as dimethylformamide,or apolar, such as dichloromethane) solvents or mixtures of solvents areused. On the other hand the therapeutic application must take place inan aqueous environment (e.g. plasma, blood, therapeutic solutions etc).In both environments (i.e. aqueous and organic solvents) the polymermatrix must be easily wettable and swellable:

(1) in order to be able to effectively remove or rinse out unbound(excess) ligand or building blocks (e.g. protected amino acids) orchemicals (e.g. coupling agents, such as carbodiimides, deprotectionagents, such as organic acids or bases), and

(2) in order to allow the respective toxin to be removed from theaqueous environment, the matrix must be wettable and swellable in thisenvironment, too.

Accordingly, the problem with known polymer matrices are that you arerestricted to the type of regenerating fluid to use in order not toelute the bound active ligand as such but only the in the materialcaught substances to be removed from the fluid, and the ligand is buildup as a defined peptide/molecule or a randomly polymerisedpolypeptid/oligomer and has then to be bound to the support material assuch—you do not know what you end up with in the end.

The advantages with the use according to the invention are that you canbuild the ligand directly on the solid support with the graftedpolyethylene glycol and the solid support with the built ligand could beused directly. This provides for reliability which you do not get withprior art matrices. Further, it provides for technological advantages asthe biologically active/-specific ligand is part of the medical deviceand the development time is shortened (this could allow or facilitate akind of individualized treatment).

During the development the material according to the invention was notknown or expected to work so brilliantly as it turned out to do. Theexpected drop backs was e.g. that polymer matrix would swell and plugthe flow through the device. Further, the pressure drop when applying afluid like whole blood was expected to be too high. Finally, as a resultof the matters above, the perfusion of the fluid through the solid phasematerial was expected to be insufficient.

The polymer matrix according to one of the preferred embodiments hasshown to be excellent for steam sterilisation and to thereafter bedried. After drying the residue of air is easily removed by adding asaline solution for the polymer matrix to swell again and being readyfor use. This leads to short preparation time in clinic, which isimportant e.g. in emergencies and in times of high workload.

Further, the material shows instant wetting, which is not the case withmost used solid supports used for different types of ligands. This is avery important feature as a solid phase material for active ligands.Even further the material is able to be compressed and decompressedwithin a certain range, and the material is biocompatible with respectto complement activation, contact phase activation, cytotoxicity andgranulocyte activation.

SUMMARY OF THE INVENTION

Therefore, the main object of the present invention is to eliminate theproblems associated with the prior art by providing a highlybiocompatible, specific, selective and easily perfused polymer affinitymatrix for removing and/or decreasing the amounts or concentrations ofthe above-mentioned undesired substances, e.g. endotoxins, in fluids,i.e. a polymer affinity matrix having all the advantages of the priorart and none of the disadvantages.

This object is achieved according to a first aspect of the presentinvention, i.e. with a polymer affinity matrix for binding one or moresubstances in a fluid for removing said substance(s) from the fluidand/or decreasing the amount or concentration thereof in said fluid witha view to preventing, eliminating, or reducing undesired activation ofcomponents or processes in said fluid, wherein said matrix comprises

a) a solid support

b) at least one spacer bound to the solid support, and, coupled to eachspacer,

c) a ligand containing at least one binding unit having at least onefunctional group, said ligand having a defined three-dimensionalstructure which is complementary as regards charge and/orhydrophobicity/hydrophilicity to the three-dimensional structure of abinding motif of said substance(s) wherein the polymer affinity matrixhas the ability to selectively bind said substance(s).

In a preferred embodiment the present invention relates to a polymeraffinity matrix for removal of endotoxins to decrease the undesiredactivation of components or processes in a fluid, wherein the matrixprovides a ligand having a three-dimensional structure complementary tothe three-dimensional structure of a binding motif of said endotoxin,thereby allowing binding of the endotoxin.

In the preferred embodiment of the polymer affinity matrix according tothe present invention the solid support is a cross-linked polystyrene,the spacer is polyethylene glycol, the at least one binding unit of theligand is an amino acid, and each functional group is an amino group orguanidino group.

In the preferred embodiment for the removal of endotoxins to decreasethe activation of the fluid, each binding unit in the polymer affinitymatrix comprises an amino acid being positively charged at or around thephysiological pH of blood, most preferably arginine, lysine, cysteine,or histidine, or another bi- or trifunctional molecule having at leastone functional group being positively charged at or around thephysiological pH of blood. Such a polymer affinity matrix generates adefined cut-off of about 1×10²-1×10⁶ Daltons and can be used for bindingboth hydrophobic and/or hydrophilic substances.

In another aspect the present invention relates to a method for removingone or more substances from a fluid and/or reducing the amount orconcentration thereof in said fluid with a view to preventing,eliminating or reducing undesired activation of components or processesin said fluid, comprising contacting the fluid with the polymer affinitymatrix according to the invention for a period of time sufficient toreduce the amount or concentration and/or remove said substance(s) ofinterest, preferably up to 24 hours, most preferably from 1 s to 2hours. However, the period depends on the flow rate, column size andmode of application, i.e. if the treatment is made in vivo, ex vivo orin vitro. Preferably, the amount or concentration of said substance(s)after having been removed or reduced is below the capacity of activatingcomponents or processes in blood or prevents activation of components orprocesses in blood.

In a further aspect the invention relates to a method for producing thepolymer affinity matrix according to the present invention, comprisingthe following steps:

-   -   a) attaching the spacer to the solid support to obtain a first        complex and    -   b) attaching to said first complex the ligand containing said at        least one binding unit with at least one functional group, or    -   c) attaching the spacer to the ligand containing said at least        one binding unit with at least one functional group to obtain a        second complex, and    -   d) attaching the solid support to said second complex, or    -   e) attaching the spacer to the solid support to obtain a first        complex, and    -   f) solid phase synthesis of the ligand on the spacer bound to        the solid support, or    -   g) building up or synthesizing the spacer from monomers directly        on the solid support by grafting, and    -   h) attaching to said first complex the ligand containing said at        least one binding unit with at least one functional group, or    -   i) building up or synthesizing the spacer from monomers directly        on the solid support by grafting, and    -   k) solid phase synthesis of the ligand on the spacer bound to        the solid support wherein information about the        three-dimensional structure, presence of charges and        hydrophobic/hydrophilic regions of the binding motif on the        substance(s) to bind is collected from X-ray crystallography,        protein sequencing, protein modelling or hydrophobicity and        hydrophilicity calculations and the binding unit is made        complementary as regards charge and/or        hydrophilicity/hydrophobicity to the binding motif of said        substance(s).

In still a further aspect the present invention relates to use of thepolymer affinity matrix for removal of one or more substances,preferably endotoxins, from a fluid, or decreasing the amount orconcentration thereof in said fluid, preferably a body fluid or atherapeutic fluid, most preferably blood.

In still a further aspect, the invention relates to a kit for removingone or more substances from a fluid and/or decreasing the amount orconcentration thereof in said fluid with a view to preventing,eliminating, or reducing undesired activation of components or processesin said fluid, said kit comprising the polymer affinity matrix, sampletubes, and a device for extra- and/or intracorporeal treatment of saidfluid, preferably blood or serum.

The use of a polymer affinity matrix according to the present inventionwill optimise the treatment of fluids, such as blood, any other bodyfluid or therapeutic fluids for removal of one or more substances, suchas endotoxins, and/or reducing the amount or concentration thereof witha view to preventing, eliminating or reducing undesired activation ofcomponents or processes in said fluid.

Specifically, the use of a highly biocompatible and perfusable materialaccording to the invention is important for prevention of the activationin the fluid during use of the polymer affinity matrix. This is ofparticular importance at extracorporeal removal of endotoxins fromplasma or blood of septic patients and is disclosed as a potentialtherapeutic strategy in the treatment of sepsis, septic shock and SIRS.As a further advantage, the use of the polymer affinity matrix accordingto the present invention will reduce the treatment time for the patient.

Further advantages and features of the present invention will becomemore apparent from the following detailed description when taken inconjunction with the drawings and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of a lipid A moiety of the LPS molecule withits structural elements.

FIGS. 2A and 2B are schematic views of examples of ligands belonging tothe tree-like structure (2A) and the comb-like structure (2B) of thepolymer affinity matrix; according to the present invention.

FIG. 2C shows examples of polymer affinity matrixes having a cyclicligand structure.

FIGS. 3A-3B show how the complementary structure of the endotoxin isgenerated by using structural requirements and positive charges,hydrophobic regions and optimal distances in the binding motif.

FIG. 4 describes different ways of producing a polymer affinity matrixaccording to the present invention.

FIG. 5 shows levels of endotoxin in human plasma after incubation withPS-PEG-beads for different time points (1, 10 and 120 minutes).

FIGS. 6A-6G show the inhibition of endotoxin induced production ofintracellular IL6 in monocytes after treatment of LPS spiked blood withPS-PEG-Arg₈ beads in comparison to beads without the matrix, i.e.reference material (PS-PEG-reference material).

FIGS. 7A-7D show the biocompatibility profile of PS-PEG-Arg₈ beadsmeasuring white blood cell (WBC) and red blood cell (RBC) numbers,thrombocyte (THR) numbers and hematocrit (HCT).

FIG. 8 shows the lack of TCC (terminal complement complex) formationafter use of PS-PEG-Arg₈ beads in hole blood.

FIG. 9 shows the absence of elastase release after se of PS-PEG-Arg₈beads in whole blood.

FIG. 10. shows the absence of TAT (thrombin-antithrombin III complex)formation after use of PS-PEG-Arg₈ beads in whole blood.

FIGS. 11A-11B show a three-dimensional structure of the ligandcontaining the binding unit of -Arg₈ and -Arg₄ optimised for LPSbinding.

FIG. 12A shows curve-fitted Langmuir isotherms for PS-PEG-Arg 8 andPS-PEG-Arg 4 related to mass of beads in gram(s).

FIG. 12B shows curve-fitted Langmuir isotherms for PS-PEG-Arg 8 andPS-PEG-Arg 4 related to moles of arginine.

DETAILED DESCRIPTION OF THE INVENTION

As indicated above, the present invention relates to a polymer affinitymatrix for removal of substances from a fluid, such as blood, any otherbody fluid or therapeutic fluids, to decrease the activation ofcomponents or processes in the fluid, while at the same time havingsufficient specificity to avoid adsorption of other valuable substancesin the fluid, e.g. physiological components of blood. Said matrixprovides a ligand having a structure that is complementary to thestructure of a binding motif of the substance to remove or reduce theamount or concentration of, e.g. an endotoxin.

Definitions

In the present context, the term “removing . . . a substance” isintended to mean preventing, reducing, decreasing, neutralising,inactivating, degrading, modifying, scavenging, binding or concealing asubstance, not always intended to mean to a zero amount or concentrationof the substance. Moreover, the term “decreasing the amount orconcentration of a substance” is intended to mean a reduction ordecrease of the amount or concentration of a substance in a fluid enoughto prevent or inhibit subsequent activation of components in the fluidand/or cellular and/or non-cellular biological mechanisms in the fluid.

With the term “preventing, eliminating, or reducing undesired activationof components or processes” the meaning is to inactivate, inhibit,lower, decrease, reduce, slow down or prevent a certain chemical,biological or biochemical process in the fluid e.g. in blood or tissuecells, e.g. plasma protein cascades such as signal transductionpathways, complement or coagulation processes and/or the activation ofcomponents involved in such processes, in a direct or indirect way. A“direct” removal of a substance influences the procedure without anyintermediate steps, whereas an “indirect” removal implies removal of asubstance being part of a long chain or cascade of reactions, whereremoval of a substance early will prevent, slow down or inhibit adownstream event of interest. This means that the removal of a substancefrom e.g. blood may give an inactivated or less activated blood. Also,implied herein is the prevention of activation of the fluid, such asblood.

The term “fluid” is intended to include any fluid, such as any gas orliquid, such as a suspension or a solution, including conventionalsolutions, e.g. aqueous, or organic solutions, or blood, any body fluidor therapeutical fluids fluids for life science applications such asfluids in biological, diagnostic or biotechnological applications e.g.buffer solutions, infusion fluids, or dialysis fluids, fluids fornutrition and fluids for industrial use.

The term “body fluid” is intended to include blood, plasma,cerebrospinal fluid, ascites and reinfusion fluids (e.g. afterhemoconcentration), blood products obtained from healthy donors, such asplasma, platelet concentrates, erythrocyte concentrates, which are usedfor transfusions.

The term “therapeutical fluid” is intended to include peritonealdialysis fluids, hemodialysis concentrates and dialysis water,substitution fluids/on line prepared fluids, infusion fluids, parenteralnutrition fluids, lavage fluids in surgical environment and fluids forblood component preparation, blood substitutes (e.g. oxygen carriers,modified hemoglobin solutions, artificial hemoglobin solutions).

The term “fluids for life science application” is intended to includefluids and/or media for cell culturing tissue engineering, molecularbiology (e.g. solutions of proteins and enzymes used for PCRtechniques), bacteriology, analytics and pharmaceutical preparation.

The term “fluids for nutrition” is intended to include drinking water,fluids for outdoor situations and reconstitution fluids for food anddrinking concentrates or powders.

The term “solid support” is intended to mean a solid phase or support oran insoluble matrix whereupon a molecule, e.g. a ligand in the form of apolypeptide, can be synthesised or coupled with or without a linker orspacer in-between.

The term “functional group” is intended to mean a specific atom, orgroup of atoms, that gives a molecule, i.e. the binding unit in thematrix according to the present invention, e.g. an amino acid, a fattyacid, a carbohydrate, a lectin, and a nucleotide, and derivativesthereof, or combinations thereof, a specific chemical characteristic orstructure, e.g. a positive or a negative charge, hydrophobicity orhydrophilicity, and/or any other physio-chemical force, e.g. van derWaals forces and π-π-interactions among aromatic groups or a capacity toform further bonds, such as hydrogen bonds or covalent bonds. Examplesof functional groups on amino acids are —COOH, —OH, —SH, guanidino, and—NH₂, but may also be a substituted amino group or any positivelycharged group or mixtures thereof.

The term “binding unit” is intended to mean the molecule mentioned aboveunder the definition of the term “functional group”, wherein saidmolecule is responsible for the binding to the substance(s) to beremoved and/or reduced, contains at least one functional group and isincluded in the ligand bound to a spacer in the polymer affinity matrixaccording to the present invention.

The term “binding motif” is intended to mean a three-dimensionalstructural and chemical motif, single or repeated, on the substance(s)to be removed.

The term “ligand” means the whole three-dimensional molecule bound tothe matrix via the spacer, i.e. comprising at least one binding unitwith at least one functional group. The ligand forms a three-dimensionalcomplementary structure with and binds to the binding motif on thesubstance to be removed. Here, “complementary” is intended to mean athree-dimensional and geometrically defined structure characterised by acapacity for precise pairing with the three-dimensional complementarystructure of a binding motif of the substance to be removed.

The term “derivatives thereof” used in connection with a certaincompound means one or more compounds derivatized in such a way that theyhave the same or essential same function as the compound as such.

Further, isomers of the compounds constituting the ligand are alsointended to be included within the scope of the invention, provided theyshow the same or essentially same function as the compounds as such. Inthe structure formulas, α and ε defines, according the nomenclaturecommonly used for amino acids, the amino groups used for covalentcoupling of further molecules.

The term “substance(s)” is intended to mean at least one component ormolecule of interest, soluble or non-soluble, which is intended to bebound to the binding unit. Examples of substances are toxic substancesthat may activate blood cells such as substances derived from virusesand bacteria, e.g. an endotoxin, a blood cell population, such as alymphocyte, thrombocyte, granulocyte, dendritic cell, monocyte,endothelial cell, stem cell, tumour cell; or a blood component or aproduct from a metabolic activity such as glucose derived molecules ordegradation products thereof, blood clotting proteins, procoagulatoryproteins, inflammatory or proinflammatory proteins, cytokines, growthfactors, hormones, chemokines, uremic toxins, and macrophage migrationinhibitory factor. Examples of blood components are infectioussubstances causing e.g. a contagious disease including virus particles,prions, or parasites, fungi, pathogen-loaded blood cells, as well asdrugs after overdosing, pathogenic food additives or other componentsnot originating from the body. Also intended to be included arepyrogens, particularly bacterial pyrogens, bacterial exotoxins, productsfrom Gram-positive bacteria, such as lipoteichonic acid, products frommetabolic disturbances, chronic or acute, as a result of e.g. diabetesmellitus, liver disease, uraemia or kidney diseases or inflammation, aswell as adhesion cascades, e.g. soluble adhesion molecules. Preferredsubstances to be removed are e.g. endotoxins from Gram-negativebacteria, such as LPS, as well as bacterial DNA or fragments ordegradation products thereof, and oligonucleotides, heparin, phosphate,blood cells, soluble or cell surface bound proteins.

The term “spacer” is intended to mean a chain-formed molecule, e.g. apolymer, which modifies the solid support in the sense of becoming apart of the solid support and positions the ligand with the at least onebinding unit containing at least one functional group away from thesolid support and makes it less restricted by steric hindrance from thesolid support and more available to the substance(s) to bind, e.g. anendotoxin, cell population or blood component. The spacer molecule maybe in a linear and/or branched and/or cyclic format. The spacer lengthis herein predefined from structural requirements of the substance(s) tobe removed.

The term “distance molecule” is intended to mean bifunctional moleculeswithin the ligand having the function of creating a structural distance,if desired, between the binding unit and the trifunctional branchingmolecule defined under general formulas I and II below and/or betweenthe spacer and said trifunctional branching molecule. The distancemolecule may also be in a cyclic format.

The term “pyrogen” and particularly “bacterial pyrogen” defines afever-producing substance, more commonly of bacterial origin, e.g. anendotoxin.

Here, the term “biocompatibility” is intended to mean the lack of orabsence of activating capacity, of e.g. activation of cells,coagulation, complement cascades or similar processes, in the sense thatthe use of the matrix does not lead to an activation of the immunesystem of the patient, or at least if such an activation occurs, it isonly to a minor degree. This means that the use of a biocompatiblecompound or substance will not lead to any unwanted or undesiredactivation of components or processes in e.g. blood or other body fluidsas well as mechanical or stress-induced cell death or cell lysis.

The Polymer Affinity Matrix

To create diversity in the polymer affinity matrix according to thepresent invention enough to bind and adsorb a large variety ofsubstances, the ligand may comprise 1-100 functional groups, preferably1-32 functional groups.

The polymer affinity matrix according to the present invention may be inthe form of a bead, a gel-like structure, a membrane or part of amembrane, a film, or a net, or a combination thereof.

Specifically, when the matrix is composed according to the presentinvention, it will generate a defined cut-off from about 1×10² to 1×10⁶Daltons and may bind both hydrophobic and hydrophilic substances withoutany restriction.

In a preferred embodiment, the polymer affinity matrix is in the form ofa PS-PEG bead (e.g. TentaGel® obtainable from Rapp Polymere Tübingen),using PEG as a spacer. PEG used as a spacer has the advantage of goodswelling of the beads in both organic and aqueous solvents and allows,due to its quasi-fluid properties, similar diffusion processes as in afluid, preferably a diffusion coefficient close to water, i.e. 40%.

The Biocompatibility

According to the invention, the polymer affinity matrix should in apreferred form show a high biocompatibility when used in a specificapplication, such as extracorporeal treatment of whole blood. A highbiocompatibility implies that certain characteristics are intended to bemore important than others. For example, no complement activation,measured by early complex formation and quantitation of terminalcomplement complex (TCC protein) according to Deppisch et al. (Kidneyint. 37:696-706) is important. Also, a low thrombogenicity is desirablefor the patient. The degree of thrombogenicity is measured byquantitation of thrombin-antithrombin III complex (TAT) according toDeppisch et al. (Neuphrol. Dial. Transplant Suppl. 3:17-23, 1994) andshould not increase during blood or plasma treatment. Still, a steadyblood cell number of white and red blood cells before and aftertreatment is included as well as an absence of cell activation due tocontact phase activation. A steady blood cell count intends to mean alow degree of or virtually no damage to blood cells due to mere stress,activation or mechanical damage.

Proteases, such as elastase produced by e.g. granulocytes orneutrophiles upon activation, are increasing in patients with bacterialsepsis and septic shock (Heiden et al. Sem. Thromb. Hemost. 1996).Neutrophilic elastase is also suggested as an early and effective markerof infection (Jensen et al., Scand. J. Clin. Lab. Invest, 1996;Groenenveld et al, Cytokine, 1995). Measurements of elastase may giveadditional information about biocompatibility, and levels shouldaccordingly not change during blood treatment.

The Ligand

To be able to create a ligand complementary to the binding motif of thesubstance to be removed, such as an endotoxin, a three-dimensionalstructure has to be formed. This may be achieved by the use of e.g. aflexible or rigid polypeptide structure, which is easy to synthesise inan optimal way for the required three-dimensional structure. Such apolymer may be linear or branched, e.g. in a tree- or comb-likestructure, or cyclic. Beneficial to the formation of suchthree-dimensional structures is the use of amino acids since theyprovide flexibility by nature. The chemistry for coupling such apolymer, e.g. a polypeptide, is well known within the art. In apreferred embodiment of the present invention such a polymer refers to apolymer containing more than one amino acid, generally up to about onehundred, i.e. an oligomer, linked together by peptide bonds. Examples oflinear polymers are poly- or oligopeptides formed by amide bonds betweenthe alpha-carboxyl and alpha-amino groups of adjacent residues, andexamples of branched polymers are poly- or oligopeptides formed by amidebonds involving one or more non-alpha-amino groups.

As stated above, the ligand molecule may be in a cyclic format. Thecyclic structure may be formed by a covalent coupling between twoappropriate functional groups within the ligand structure, e.g. adisulfide bond (—S—S—) between two SH groups of cysteine by oxidation (acommon cyclisation reaction also occurring in natural proteinstructures).

FIGS. 2A and 2B show examples of different branched, tree-like andcomb-like structures, respectively, of the ligand coupled to a spacerbound to a solid support. In these structures the ligands are basicallybuilt up of lysine residues, and the binding units in each ligand arearginine residues. The ligand included in the polymer affinity matrixaccording to the present invention may be represented by the twofollowing formulas:—X¹ _(n)—Y_(m)[X² _(i)-Z¹; X³ _(j)-Z²]_(1/2 (m+1))  General Formula I:

In general Formula I, representing a tree-like structure, the differentsymbols have the following meaning:

n=0 or 1;

m=2^(k)−1;

k=0 to 10, wherein if k 0 then X₂=X₃ and Z₁=Z₂;

i=0 or 1; and

j=0 or 1,—(X¹ _(n)—Y¹[Y² _(m)[X² _(i)-Z¹; X³ _(j)-Z²]_(1/2(m+1)))_(r)—X⁴_(p)-Z³  General Formula II:

In general Formula II, representing a comb-like structure, the differentsymbols have the following meaning:

n=0 or 1;

m=2^(k)−1;

k=0-10, wherein if k=0 then X₂=X₃ and Z₁=Z₂;

r=1-100;

i=0 or 1;

j=0 or 1; and

p=0 or 1.

Z¹, Z² and Z³ each represents the binding unit and is each an organicmolecule chosen from the group consisting of an amino acid, a peptide, afatty acid, a carbohydrate, a lectin, and a nucleotide, and derivativesthereof, or combinations thereof, wherein Y, Y¹ and Y² each is atri-functional branching molecule which contains functional groupschosen from the group consisting of amino, hydroxy, aldehyde,isocyanate, isotiocyanate, thiol, maleimido, epoxy, and derivativesthereof, or combinations thereof, and wherein

X¹, X², and X³ each is an optional bifunctional distance moleculecontaining two functional groups chosen from the group consisting ofamino, carboxy, hydroxy, aldehyde, isocyanate, isothiocyanate, thiol,maleimido, epoxy, and derivatives thereof, or combinations thereof.

In FIG. 2C examples of polymer affinity matrixes having a cyclic ligandstructure are shown. In the structure formulas R means-Lys_(m)[Arg]_((m+1)), wherein m=2^(k)−1, and examples wherein k=0, 1,and 2 are shown.

Thus, the at least one binding unit of the ligand in the polymeraffinity matrix according to the invention contains at least onefunctional group, preferably an amino group or an guanidine group or asubstituted amino group or at least one of said functional groups.

Preferably, the amino acids are chosen for their characteristic of beingpositively charged at or around physiological pH, specifically of blood≧7.2, i.e. at a pK about ≧6.0. Examples of such preferred amino acidsare arginine (Arg), lysine (Lys), and histidine (His). Also mixtures ofsaid amino acids may be used in the ligand of the matrix, to createstill a further variability.

Most preferably, the ligand comprises arginine as binding unit(s) andconstitutes ≦3 mmol/g matrix. In general, the amount of amino acids maybe at least about 0.01-5 mmol/g matrix.

In the embodiment when the binding unit of a branched ligand comprisesthe amino acid arginine, the number of arginine molecules per ligand is2-100 arginine molecules, and preferably 4-8 molecules, known as Arg₄₋₈.Still, by using a trifunctional amino acid, i.e. containingthree-functional groups, here two amino groups and one carboxy group,such as lysine, a branched structure can be created. This approach ofusing so called multiple antigenic peptides, MAP, has been introduced byTam et al., and is described in U.S. Pat. No. 5,229,490. By variation ofthe number of branches, the clustering and the distances of the endgroups, e.g. positively charged arginine comprising terminal positivefunctional groups, can be varied, as discussed above in connection withFIG. 2.

In a specific embodiment of the invention, where endotoxins are thesubstances to be removed by the polymer affinity matrix, the positivecharges of the functional groups of the amino acids are held at adistance defined by the distance between individual negatively chargedphosphate groups in an endotoxin as shown in FIG. 3.

As discussed above, the amount and the configuration of the amino acidscreate the variability of the complementary ligand in the polymeraffinity matrix, and may therefore vary to create sufficient variabilityfor absorbing a large variety of substances. In another embodiment ofthe invention the amount of amino acid is at least about 0.01, 0.1, 1,2, 3, 4, or 5 mmol/g matrix.

Design of the Ligand Containing the Binding Unit(s)

To achieve a complementary structure to a substance, information iscollected from procedures known in the art for studying therelationships regarding structure, presence of positive charges beingheld at a certain distance as well as presence of a hydrophobic regionin proximity to the charged groups, e.g. X-ray crystallography, proteinsequencing, protein modelling or hydrophobicity and hydrophilicitycalculations as described in Example 1 and shown in FIGS. 3 and 11.

In the preferred embodiment, the substance to be removed is anendotoxin, such as LPS containing lipid A, and information known to theskilled man in the art about the molecule regarding e.g. structure anddistances between charged phosphate groups within the molecule is usedto form a ligand with at least one binding unit in the polymer affinitymatrix. The polymer forming the ligand is in this embodiment synthesisedby amino acids, e.g. arginine and/or lysine. However, other amino acidsmight be used, such as histidine or cysteine.

In the Arg₄₋₈ embodiment of the invention as the complementary part tothe binding motif of the substance to remove, e.g. an endotoxin, saidarginine as binding units possess the following properties for theremoval of the endotoxins as shown in FIG. 3:

-   -   a) presence of positive charges being held at a distance which        optimally fits with the distance of the negatively charged        phosphate group in lipid A,    -   b) presence of a hydrophobic region in proximity to the charged        groups allowing hydrophobic interactions with the fatty acid        moieties of lipid A, and    -   c) a combination of said properties in a) and b) to give a        complementary structure to lipid A allowing optimal binding.        The Spacer

The polymer affinity matrix according to the invention comprises aspacer that is substantially hydrophobic or hydrophilic, modifying thesolid support in the sense of becoming a part of the solid support, andhas the function of an anchoring part for the ligand comprising the atleast one binding unit with at least one functional group.

Preferably, polyethyleneglycol (PEG) is used as spacer molecule, in alinear or branched configuration at the preferred average molecularweight of 400-10 000 Daltons and is represented by the formulaH—(OCH₂CH₂)_(n)—OH, wherein n is about 2-250. The flexibility of PEGchains allows a good access of toxin molecules within thethree-dimensional structure of the ligand polymer.

Moreover, in another embodiment of the present invention the solidsupport may be provided with two or more spacers of the same ordifferent kind, e.g. polyethyleneglycols, polypropyleneoxides,polyvinylalcohols, polyvinylamines, polyglycidoles, orpolyethyleneimines, and derivatives thereof.

Alternatively, the presence of a spacer in the polymer affinity matrixis not required, e.g. when the three-dimensional structure of thecomplementary binding motif of the substance(s) to bind allows theabsence of the spacer for the binding to occur. Thus, according to thisembodiment of the present invention the binding unit is directly boundto the solid support.

The Solid Support

The solid support should provide variability in porosity and sizeexclusion characteristics. Also, it should provide a support for solidphase synthesis for polypeptide synthesis in the preferred embodiment.Different solid supports, i.e. polymers, that may be used in the presentinvention are described in EP 0 187 391, WO 99/17120, and the U.S. Pat.No. 4,908,405. Here, the solid support is a linear and/or branchedbiocompatible graft copolymer having a degree of cross-linking of0.05-10% and is selected from the group consisting of polyvinylalcohols,polyhydroxystyrenes, polymers produced from chloromethylatedpolystyrenes and ethylene glycols; poly- or oligoethylene glycols of theformula H—(OCH₂CH₂)_(n)—OH, where n represents 2 to 250, polyacrylatesor polymethacrylates functionalised with hydroxy groups; and derivativesthereof. In the present invention the above-mentioned degree ofcross-linking may be up to 50%.

The solid support material is selected from a group consisting of,besides the above-mentioned, polystyrene, hydroxyalky-polystyrenes,hydroxyaryl-polystyrenes, hydroxyalky-aryl-polystyrenes,polyhydroxyalkylated polystyrenes, polyhydroxyarylated polystyrenes,isocyanatoalkyl-polystyrenes, isocyanatoaryl-polystyrenes,carboxyalkyl-polystyrenes, carboxyaryl-polystyrenes,aminoalkyl-polystyrenes, aminoaryl-polystyrenes, cross-linkedpolyethyleneglycols, polyacrylates, polymethacrylates, cellulose,silica, carbohydrates, latex, cyclo-olefine copolymers, glass, othersuitable polymers or combinations thereof. The form of the solid supportmay be a bead, membrane, particle, e.g. a nanoparticle, net, woven andnon-woven fabrics, fibre mat, tube, film, foil or combinations thereof,or cross-linked interpenetrating networks. In a preferred embodiment;the affinity matrix described above consists of cross-linked polystyreneonto which PEG as a spacer is grafted.

A preferred solid support material in the present invention is a bead,having a size sufficient to provide a highly perfusable andbiocompatible support, e.g. polystyrene, preferably in the combinationwith PEG as a spacer to which lysine and/or arginine is attached, whichshould have no restrictions for hydrophilic or hydrophobic substances. Alist of suitable commercially available beads is shown in Table 1. TABLE1 Commercially available activated beads^(a) Toyo Pearl HW70EC ®(TosoHaas) Toyo Pearl HW65EC ® (TosoHaas) Toyo Pearl AF650M ® (TosoHaas)TentaGel ® (Rapp Polymer) Eupergit C250L ® (Röhm) Eupergit 250 ® (Röhm)Fractogel EMD Epoxy ® (M) (Merck) Fractogel EMD Azlactone ® (S) (Merck)Poros EP ® (Perkin Elmer-Biosystems)^(a)The commercially available activated beads above can be conditionedfor immobilization of a ligandPerfusability of the polymer affinity matrix

A hydrogel like structure is formed when the polymer affinity matrix ishydrated and, due to the hydration, swells. The swelling capacity isdefined as the swelling due to hydration of the polymer from a dry stateto form the hydrated and gel-like matrix. Such a swelling may be definedas an increase in weight per volume unit when hydrated and it mayaccording to the invention be a factor of about 1.5-10 times, preferably3-5 times, when comparing dry and hydrated forms of the polymer affinitymatrix. This swelling capacity allows whole blood to perfuse completelythrough the matrix, still keeping the blood cells and numbers intact. Asstated above, in the preferred embodiment, the polymer affinity matrixas such generates a matrix with a defined cut off of about 1×10²-1×10⁶Daltons allowing blood cells to perfuse and with almost no diffusionalrestriction for hydrophilic and/or hydrophobic substances.

Method for Removing a Substance

The present invention also relates to a method for removing one or moresubstances from a fluid and/or reducing the amount or concentrationthereof in said fluid with a view to preventing, eliminating or reducingundesired activation of components or processes in said fluid,comprising contacting the fluid with the polymer affinity matrixaccording to the present invention for a period of time sufficient toreduce the amount or concentration of and/or remove said substance(s).In a preferred embodiment using this method, the removal of a substance,e.g. an endotoxin from blood or any other body fluid, will therebydirectly or indirectly prevent activation of e.g. blood cells by bindingto the undesired substances listed under the definition of“substance(s)” above. Preferably this may be achieved by contacting thefluid with the polymer affinity matrix defined above for a period of upto 24 hours, most preferably from 1 s to 2 hours, giving a lessactivated or prevented activation of blood and its components.

In a preferred embodiment, the substance to be removed according to themethod disclosed in the present invention is an endotoxin. The presentinvention also relates to removal of a defined blood cell type orpopulation selected from leukocytes, e.g. T and B cells, monocytes,thrombocytes, granulocytes and/or neutrophiles. Also intended to beremoved by the above described polymer matrix are components in anextra- or intracellular signalling transduction pathway selected fromthe group consisting of glucose and its degrading products, carbonylcompounds, inflammatory and proinflammatory proteins and/or proteinsinvolved in thrombogenesis or in the complement cascade, bacteriaderived constituents, endotoxins, cells, blood cells, bacteria andviruses, or pathogen-loaded blood cells, or at least parts ordegradation products thereof, DNA or fragments thereof, or phosphate, byproviding a ligand comprising at least one binding unit and having astructure which is complementary to the structure of a binding motif ofthe substance.

In this preferred embodiment, an endotoxin, e.g. LPS, is removed to <50%over two hours during e.g. a static incubation or during perfusion of asolid phase column.

In this preferred embodiment, the endotoxin, is removed during a definedtime period of about 1 second to about and including 2 hours to anamount or concentration inactivating the blood or preventing activationof the blood, as measured according to an LAL assay, which is describedbelow. Using the LAL assay is known in the art and will provideinformation about the endotoxin levels. The method according to theinvention, which is fast and efficient, will yield levels of theendotoxin according to the LAL assay, within the above mentioned timeperiod of 1 s-2 h, below the capacity of activating blood, or onlyactivating blood cells and components to a minor degree.

Method for Producing a Polymer Affinity Matrix

The method for producing a polymer affinity matrix according to thepresent invention comprises the following steps:

-   -   a) attaching the spacer to the solid support to obtain a first        complex, and    -   b) attaching to said first complex the ligand containing said at        least one binding unit with at least one functional group; or    -   c) attaching a spacer to the ligand containing said at least one        binding unit with at least one functional group to obtain a        second complex, and    -   d) attaching the solid support to said second complex; or    -   e) attaching the spacer to the solid support to obtain a first        complex, and    -   f) solid phase synthesis of the ligand on the spacer bound to        the solid support, or    -   g) building up or synthesizing the spacer from monomers directly        on the solid support by grafting, and    -   h) attaching to said first complex the ligand containing said at        least one binding unit with at least one functional group, or    -   i) building up or synthesizing the spacer from monomers directly        on the solid support by grafting, and    -   k) solid phase synthesis of the ligand on the spacer bound to        the solid support,        wherein information about the three-dimensional structure,        presence of charges and hydrophobic/hydrophilic regions of the        binding motif on the substance(s) to bind is collected from        X-ray crystallography, protein modelling or hydrophobicity and        hydrophilicity calculations and the ligand containing the        binding unit is made complementary as regards charge and/or        hydrophilicity/hydrophobicity to the binding motif of said        substance(s).

The method described above also includes in one embodiment theproduction of a matrix with a spacer molecule comprising a ligandimmobilised on a solid support according to any of the following ways asshown more in detail in FIG. 4;

for a) and b) above: activation of the solid support, coupling of thespacer molecule to the solid support, synthesis of the ligand containingthe binding unit, and site specific coupling of the ligand to the spacermolecule, or

for c) and d) above: synthesis of the ligand containing the bindingunit, coupling of the spacer molecule to the ligand, activation of thesolid support, and site specific coupling of the spacer-ligand complexto the activated solid support, or,

for e) and f) above: activation of the solid support, coupling of thespacer molecule to the activated solid support, and solid phasesynthesis of the ligand containing binding unit on this support.

The method containing steps, a)-f) described above also includes in asecond embodiment the specific steps of, for a) and b), activation ofthe spacer, coupling of the activated spacer to the solid support, andcoupling the ligand to said activated spacer, or,

for c) and d), synthesis of the ligand, activation of the spacer, sitespecific coupling of the ligand to the activated spacer molecule andcoupling of the spacer-ligand complex to the solid support, or,

for e) and f), activation of the spacer, coupling of the activatedspacer to the solid support and solid synthesis of the ligand on thespacer bound to the solid support.

As stated above, preferred by the present invention is the method above,wherein the solid support, the spacer and the ligand are immobilised byactivation of a solid support, coupling of the spacer molecule, andsolid phase synthesis of the ligand containing the binding unit directlyon the solid support.

Use of the Polymer Affinity Matrix

The present invention also relates to the use of a polymer affinitymatrix according to the present invention, preferably for use forremoval of one or more substances, preferably endotoxins, from a fluid,or decreasing the amount or concentration thereof in said fluid,preferably a body fluid or a therapeutic fluid, most preferably blood,in particular for the production of less activated blood or preventionof undesired activation of components or processes in blood. The polymeraffinity matrix is preferably used as a part in an extracorporeal bloodpurification process or in contact with blood or a blood stream, e.g. asan implant in the body to contact blood or any body fluid, e.g. thevascular system, blood vessels or in the peritoneal cavity.

Kit Comprising the Polymer Affinity Matrix

In one embodiment the present invention refers to a kit for removing oneor more substances from a fluid and/or decreasing the amount of orconcentration thereof in said fluid with a view to preventing,eliminating, or reducing undesired activation of components in saidfluid, said kit comprising a polymer affinity matrix according to thepresent invention, sample tubes, and a device for extra- and/orintracorporeal treatment of said fluid, preferably blood or serum.

Conclusion

The present invention provides means for the removal of substances froma fluid in a highly efficient and time-saving way. This is achieved bythe use of a polymer affinity matrix according to the invention that ishighly biocompatible and allows for whole blood to flow through due togood swelling capacity. The efficient means are also due to thegeneration of a ligand comprising a binding unit complementary to thebinding motif of the substance to be removed. Therefore, the presentinvention may be applied in e.g. extracorporeal blood treatment such asdialysis and transfusion medicine for therapeutic applications, stemcell therapy and/or therapeutic cell therapy, diagnostic applicationsand also in biotechnology, bioengineering, gene technology, foodchemistry and water preparation and/or purification.

The present invention also concerns the use of a polymer matrix for theproduction of a polymer affinity matrix for removal of one or moresubstances from a fluid or decreasing the amount or concentrationthereof in said fluid, wherein the specific affinity is dependent on anyligand applied on the polymer matrix, wherein the polymer matrixincludes a solid support and a spacer, wherein the solid support is madeof a material selected from the group consisting of polystyrene,polyvinyl alcohols, polyhydroxystyrenes, polymers produced fromchloromethylated polystyrenes or polyacrylates, polymethacrylatesfunctionalised with hydroxy groups, hydroxyalkyl-polystyrenes,hydroxyaryl-polystyrenes, hydroxyalkyl-aryl-polystyrenes,polyhydroxyalkylated polystyrenes, polyhydroxyarylated polystyrenes,isocyanatoalkyl-polystyrenes, isocyanatoaryl-polystyrenes,carboxyalkyl-polystyrenes, carboxyaryl-polystyrenes,aminoalkyl-polystyrenes, aminoaryl-polystyrenes, polymethacrylates,cross-linked polyethyleneglycols, cellulose, silica, carbohydrates,latex, cyclo-olefine copolymers, glass or combinations thereof,preferably a cross-linked polystyrene, and wherein the spacer isselected from the group consisting of poly- or oligoethylene glycols ofthe formula H— (OCH₂CH₂)_(n)—OH, wherein n represents 2-250.

In a preferred embodiment of the invention the solid support has theform of a bead, gel, membrane, particle, net, woven or non-woven fabric,fibre mat, tube, film, foil or combinations thereof or cross-linkedinterpenetrating networks.

In another preferred embodiment of the invention the spacer is apolyethylene glycol (PEG) in a linear and/or branched configuration andhas an average molecular weight of 400-10 000 Daltons, or derivativesthereof.

In another preferred embodiment of the invention the polymer matrix hasa swelling capacity enough to allow perfusion of plasma or whole blood.

In another preferred embodiment of the invention the swelling capacityof the polymer matrix is about 1.5-20 fold, preferably 2-6 fold, from adry state to the hydrated form.

In another preferred embodiment of the invention the polymer matrix hasthe form of geltype beads.

In another preferred embodiment of the invention said fluid is anaqueous or organic solution, a body fluid, preferably blood, therapeuticfluids, fluids for life science applications, preferably buffersolutions, infusion fluids or dialysis fluids in biological, diagnosticor biotechnological application, blood products obtained from healthydonors, such as plasma, platelet concentrates, erythrocyte concentrates,preferably oxygen carriers, modified hemoglobin solutions and artificialhemoglobulin solutions, fluids for nutrition and fluids for industrialuse.

In another preferred embodiment of the invention the polymer matrix hasa cut-off value of from about 1×10² to about 1×10⁶ Daltons and bindshydrophobic and/or hydrophilic substances.

In another preferred embodiment of the invention the solid support is across-linked polystyrene, the spacer is a polyethylene glycol.

The rational development of specific binding structure applicable inextracorporeal blood treatment requires: (i) a flexible technology forbuilding up ligands and (ii) basic requirements on biocompatibility,toxicology and processability. Solid phase peptide synthesis is appliedto obtain well defined ligand structures assembled on biocompatiblepolymer substances, which is, according to one of the preferredembodiments, polystyrene-polyethylene glycol grafted copolymers specificfor biologically substances.

By systematic variation of the geometrical structure of ligand motifs wecould demonstrate that an increase in ligand affinity by a factor of 10to 100 relative to the molar concentration of the building blocks. Theseinvestigations were carried out in human serum, i.e. in the presence ofcompetitive proteins.

The assessment of blood compatibility was done by in vitro assays inhuman plasma and/or whole blood. The PS-PEG base material with orwithout the ligand is biocompatible with respect to complementactivation, contact phase activation, cytotoxicity and granulocyteactivation.

The base polymer structure shows an advantageous biocompatibilityprofile especially for extracorporeal application. The appliedtechnology for ligand synthesis allows the processing of specificpolymer materials without generation of toxic residuals.

EXAMPLES Example 1

Design of the Ligand Containing a Binding Unit for LPS

This example describes, without limiting the invention, the design of aligand containing a binding unit for the removal of LPS.

Principle

An efficient removal of toxins, e.g. LPS, requires a ligand with abinding unit that is complementary to the binding motif of the substanceto be removed. This includes a three-dimensional aspect of the substanceto be removed and a precise arrangement of molecules to optimise abiospecific recognition with characteristics like complementary charges,hydrophobicity and hydrophilicity. This, together with appropriatedistances of the aforementioned characteristics, will complete theligand with its binding unit. Also, the total three-dimensionalpresentation of such a ligand within a polymer matrix should be optimalin space for a high perfusion, ligand presentation and flexibility ofthe ligand. Still, a high biocompatibility is a prerequisite for in orex vivo blood purification in patients. In the described matrix, theaccessibility is comparable to or even identical to a non-solid phaseaqueous solution. The structure of LPS is shown in FIG. 1. The suggestedcomplementary binding to LPS is shown in FIGS. 3A-D, wherein thephosphate groups and hydrophobic tail of LPS are considered for theformation of a complementary binding motif. The suggested structure isshown for Arg₄ and Arg₈ in FIG. 2 as well as in a three-dimensionalformat in FIG. 11. The Arg₈ shows half of the arginine positions in theleft handed Figure and half of the arginine positions in the right handFIG. 11B. Also, the link to the spacer, here to PEG, is shown.

Molecular Simulation

Molecular simulations were further applied to identify and verify thethree-dimensional geometric and chemical structure of the describedbinding site. The simulations were performed on a Silicon GraphicsOctane2 Workstation, using the Insight II-Package (Molecular SimulationsInc. (MSI), San Diego, Calif.) and the optimisation algorithms includedherein, e.g. AMBER-forcefields. The results of the tree-dimensionalarrangements for Arg₄ and Arg₈ are shown in FIGS. 11A and B,respectively. Here, the illustration shows the principle of thethree-dimensional terminal part of the matrix, without the PEG spacer orsolid support.

Synthesis of the Ligand

The ligand with its binding unit is synthesised according to FIG. 4,wherein three different way are illustrated. A preferred method of thethree is solid phase synthesis directly onto the PEG spacer attached tothe solid support. The binding unit is used herein in further examples.

Example 2

Comparison of Branched and Linear Ligands

This example describes the adsorption of the endotoxin LPS from humanplasma. The example also shows a comparison between linear and branchedligands for the adsorption of the endotoxin.

Principle

Beads are incubated with heparinised human plasma for a time period oftwo hours. The endotoxin levels are determined after two hours ofincubation using an LAL assay.

Material

The following beads are used: PS-PEG-LBP 94-108 endotoxin-bindingsequence from LBP, linear PS-PEG-BPI 85-99 endotoxin-binding sequencefrom BPI, linear PS-PEG-Arg₈ 8-fold branched containing argininePS-PEG-NH-Ac acetylated base-material as control No beads ControlProcedure

Incubation of beads in heparinised plasma is carried out at 37° C.Samples are slowly agitated and beads are removed by centrifugationafter an incubation period of two hours.

Analysis

An LAL assay is performed to quantitate the levels of endotoxins afterthe incubation with beads. The assay is performed by an LAL inducedchromogenic substance reaction (Chromogenics, Mölndal, Sweden). Levelsare calculated according to a standard curve obtained with definedconcentrations of endotoxin in heparinised human plasma.

Results

Using the LAL assay the following endotoxin concentrations were foundafter a two-hour incubation period. Endotoxin (IU)/ml PS-PEG-LBP 94-10810 PS-PEG-BPI 85-99  9 PS-PEG-Arg₈  2 PS-PEG-NH-Ac 10 No beads  8Starting value 10Discussion

This experiment shows that the endotoxin levels could be decreased5-fold by using the branched, three-dimensional matrix onto thePS-PEG-beads. Beads with a linear ligand were not as efficient andendotokin levels in those samples were still at, or near, startinglevels of the endotoxin.

Example 3

Kinetics of Endotoxin Binding

This example shows the kinetics of endotoxin binding when beads with athree-dimensional matrix, optimised for LPS binding, are incubated withplasma and analysed at different points of time.

Principle

The kinetics of absorption is dependent on the structure and the degreeof cross-linking of the polymer matrix. Beads are incubated withheparinised human plasma. After 1, 10, and 120 minutes samples arewithdrawn. The endotoxin levels are determined using an LAL assay.

Material

The following beads are used: PS-PEG-Arg₈ 8-fold branched containingarginine PS-PEG-Arg₄ 4-fold branched containing arginine PS-PEG-Arglinear containing arginine PS-PEG-NH-Ac acetylated base material (-Ref)Procedure

Incubation of beads in heparinised plasma is carried out at 37° C.Samples are slowly agitated and test samples are withdrawn after anincubation period of 1, 10, 120 minutes. Beads are removed bycentrifugation.

Analysis

An LAL assay is performed for the quantification of endotoxin levels asin Example 2.

Results

FIG. 5 shows the results of the endotoxin levels measured at differentpoints of time. The Figure shows that a time period of two hours isneeded for efficient removal, i.e. yielding 20-30% endotoxin left of thestarting levels in the samples. The linear ligand containing arginine(PS-PEG-Arg) is only removing half the amount of endotoxins, i.e. alevel of 60% left after 120 minutes. Similarly, where the referencebeads (PS-PEG-NH-Ac) were added, the amount of endotoxin afterincubation with the beads was still unchanged, i.e. at starting values,after 120 minutes.

Example 4

Influence of the Terminal Amino Acid and Branching of the Polymer

This experiment shows the influence of the terminal amino acid and(arginine vs lysine) and the influence of branching, non-branched v.s.4-fold v.s. 8-fold branched, of the ligand.

Principle

Beads are incubated with heparinised human plasma with or withoutendotoxin. The endotoxin levels are determined using an LAL assay aftertwo hours.

Material

The following beads are used: PS-PEG-Arg₈ 8-fold branched containingarginine PS-PEG-Lys₈ 8-fold branched containing lysine PS-PEG-Arg₄4-fold branched containing arginine PS-PEG-Lys₄ 4-fold branchedcontaining lysine PS-PEG-NH-Ac acetylated base material PS-PEG-Arglinear containing arginine No beads ControlAnalysis

An LAL assay is performed as in Example 2.

Results Endotoxin (IU/ml) PS-PEG-Arg₈ 0.4 PS-PEG-Lys₈ 1.8 PS-PEG-Arg₄0.01 PS-PEG-Lys₄ 2.7 PS-PEG-NH-Ac 7.8 PS-PEG-Arg 3.5 No beads 11 Beforeincubation, i.e. 13 starting valuesDiscussion

The results show that a branched ligand is more efficient than linearforms, and that arginine is several-folded (4-folded and 200-folded forArg₈ and Arg₄ respectively) more efficient than lysine in the removal ofendotoxins. Also, the PS-PEG-Arg₄ is the most efficient in the removalof endotoxins after two hours.

Example 5

Reduction of Endotoxin Dependent IL6 Induction in CD14+Monocytes

This example describes the reduction of endotoxin dependent IL6induction in human monocytes by the use of PS-PEG-Arg₈.

Principle

In response to LPS, monocytes start to transcribe and express IL6. Thelevels of upregulated IL6 can already be measured after 4 h byintracellular flow cytometry analysis (FACS).

Material

Human mononuclear cells from whole blood are used. PS-PEG-Arg₈ is usedfor the removal of endotoxin and PS-PEG-NH-Ac is included as a control.Lipopolysaccaride (E. coli strain 055:B5 from Biowittaker Co.) is usedfor stimulation of the MNC in vitro. FACS analysis is performed using aFACSCAN Calibur (Beckton Dickinson®)

Procedure

Whole blood is incubated at 37° C. (5%° CO₂) for 30 minutes with 10 or30 IU/ml LPS. Samples are incubated in parallel with and withoutPS-PEG-Arg₈ beads, as well as control beads PS-PEG-NH-Ac. The cells arefixed in PermFix (Beckton Dickinson®) and a double staining performedwith anti-CD14-FITC and anti-IL6-PE. 20 000 cells are counted per cellsample for FACS analysis. Monocytes defined as CD14⁺ cells normallyconstitute about 5% of the total cell population.

Analysis

Monocytes are defined as CD14+cells in the cell suspension.Intracellular IL6 (icIL6) is calibrated to an internal standard andquantitated in the CD14⁺ cell population using FACS analysis after theincubation with 10 or 30 IU/ml LPS for 30 minutes. Calculations areperformed using CellQuest Software Package (Beckton Dickinson®)

Results

Using the FACS data and calculations performed therefrom, analysis by askilled man in the art of the CD14⁺ monocyte population generates thefollowing data displayed in FIG. 6. Shown in the graph is a 70%reduction of the endotoxin levels when using PS-PEG-Arg₈ beads comparedto the control beads (PS-PEG-NH-Ac) and samples with no beads included.The intracellular levels of IL6 are shown in histograms. On the righthand side, a decrease can be seen in IL6 levels after incubation withPS-PEG-Arg₈ beads. The lower left row shows the absence of intracellularIL6 in fresh cells.

Discussion

This experiment shows a rapid and complete inhibition of LPS stimulationin a human CD14⁺ monocyte population measured by an inhibition of theLPS dependent IL6 upregulation in monocytes.

Example 6

Cell Counts after Whole Blood Perfusion

This example describes, without limiting the invention, thepermeabilisation of blood cells after perfusion through the polymeraffinity matrix on beads.

Principle

Blood purification requires no damage to or activation of cells.Mechanical damage, especially of erythrocytes, could lead to haemolysisand subsequent life-threatening complications. A hydrogel like polymermatrix according to the invention can surprisingly be perfused by wholeblood cells.

Material

A highly swellable polymer matrix, PS-PEG-Arg₈ beads, are used. Columns,ID 20 mm, are filled with 1 g of the matrix, absorbing 4-5 ml water andsubsequently prerinsed with physiological saline solution before use.The columns are then used for perfusion of whole blood.

Procedure

Fresh whole blood with citrate and LPS (30 IU/ml) is perfused throughcolumns using sterile equipment in a laminar flow. LPS-spiked wholeblood is perfused at a flow of 5 ml/min at 37° C. Blood samples weredrawn before and after perfusion and analysed. Cells analysed are redblood cells (RBC), hematocrit (HTC) and free hemoglobin, white bloodcell (WBC), platelets, and thrombocyte numbers (TRC).

Cell number is counted pre- and post column perfusion by a CoulterCounter® (Becton Dickinson).

Free hemoglobin is measured as a total hemoglobin level after a completelysis of erythrocytes followed by a photometric quantification at 405nm. Integrity of erythrocytes after whole blood perfusion is shown bymeasurements of free hemoglobin in plasma by a photometricquantification at 405 nm.

Results and Discussion

Cell numbers and HCT are measured and the results are shown in FIGS. 7A(thrombocytes), 7B (HCT), 7C (RBC), and 7D (WBC). In addition, noincrease in free hemoglobin is found, i.e. no haemolysis is induced(data not shown). Data from this experiment shows that, after theinitial transient retention time due to an initial dilution effect, thecell number is stabilising to pre-column values. The data shows apermeabilisation of the matrix material of about 100%, since thepre-column cell count is the same as the post-column cell count. Also,the cells keep intact, as measured by viable cell count and red bloodcell lysis.

Example 7

Biocompatibility Features

This example describes parts of the biocompatibility profile of thePS-PEG-Arg₈ matrix.

Principle

Biocompatibility or non-compatibility is herein measured as complementactivation, elastase release, and trombogenicity bythrombin-antithrombin III complex formation.

Material

As in Example 6, cellulosic material is used as a reference.

Method

Elastase is measured in plasma from whole blood perfusion using aspecific enzyme linked immunosorbent assay (ELISA; Diagnostic ProductCo.).

Complement activation is measured by quantitation of terminal complementcomplex (TCC protein) is measured according to Deppisch et al. (KidneyInt. 37:696-706).

TAT, the degree of thrombogenicity, is measured according to Deppisch etal. (Neuphrol. Dial. Transplant Suppl. 3:17-23, 1994).

Results

In FIG. 8, the formation of TCC is shown over time. Reference beads,PS-PEG-NH-Ac (TG-base or -ref material) shows a 4-5 fold increase in TCCformation. After an initial rise over 10-15 min. for PS-PEG-Arg₈ the TCCformation stabilises at pre-perfusion levels.

In FIG. 9, the level of elastase is measured over time. Levels are notchanging compared to pre-column values. Measurements over 35 minutes areshown. For the reference material the elastase levels are increasing 8-9fold over 35 minutes.

In FIG. 10, the formation of TAT is shown. No increase in TAT formationis detected.

Discussion

Biocompatibility features are important in ex vivo and in vivo bloodtreatment, e.g. dialysis. The described matrix shows no activation ofthe complement system, no activation of the coagulation system and noelastase release compared to the reference material included, i.e. ahigh biocompatibility. This, together with the high perfusion capacityof whole blood as shown in Example 6, shows that the polymer affinitymatrix according to the invention is highly suitable for extracorporealtreatment of blood, whole blood included.

Example 8

Further Endotoxin Adsorption Experiments

Endotoxin adsorption experiments have been performed in order to showthe importance of finding a specific geometrically defined arrangementof functional groups (i.e. arginine residues) which build up a ligandfor endotoxin binding. It is shown that the endotoxin binding does notjust depend on the amount of arginine (i.e. the positive charges) butthat the defined geometrical arrangement is more important.

Procedure

Human serum (as a representative of human body fluids such as blood,plasma etc) was spiked with defined amounts of endotoxin (i.e. 3 IU/mland 10 EU/ml) and incubated with 40 mg of PS-PEG-Beads containing eitherthe Arg8 arrangement or the Arg4 arrangement as ligand. The two kinds ofbeads/ligand arrangements contain different amounts of arginine: Arg4contains 0.72 mmol/g and Arg8 contains 1.89 mmol/g. The higher argininecontent of the Arg8 beads is a consequence of the additional branchingcaused by the lysine bifurcations. After 30 min of incubation the free(i.e. not bound to the surface or matrix) concentration of endotoxin wasmeasured (by LAL test) in the supernatant serum.

Analysis TABLE 2 Results of the incubation experiment with human serumspiked serum before incubation 0 3 10 EU/ml serum no beads 0 3   9.1EU/ml after 30 min PS-PEG-Arg4 0 1.5 5.5 EU/ml incubation PS-PEG-Arg8 00.2 1.6 EU/ml

These data were analyzed by using the Langmuir approach which describesligand-receptor interactions and equilibria at solid surfaces (e.g.protein adsorption) (Ref: J D Andrade: Principles of protein adsorptionin: J D Andrade (Ed.) Surface and Interfacial Aspects of BiomedicalPolymers. Volume 2, Protein Adsorption, Plenum Press New York 1985, pp1-80.). This analysis is done by plotting the amount of toxin (i.e.endotoxin) bound to the ligand-matrix (calculated from the differencebetween amount added and amount left in the supernatant afterincubation) divided by the total number of ligands present at the matrixversus the equilibrium concentration of the toxin (i.e. the endotoxinconcentration after incubation) in the supernatant. The plots are thenfitted to the mathematical expression of the Langmuir isotherm, whichgives the affinity or association constants for the differentmatrix-bound ligands towards the toxin. In FIGS. 12A and 12B examples ofthe Langmuir approach are shown.

Two kinds of Langmuir plots were performed, which differ by the way thetotal number of ligands present at the matrix is defined:

(1) neglecting the different arginine content of the beads, i.e. by justtaking the mass of beads in grams as a measure of the total number ofligands (FIG. 12A); this approach is justified since in the practicalapplication of the ligand-polymer-matrix, e.g. in an adsorber device tobe perfused with a human body fluid such as blood or plasma in anextracorporeal circuit, it is to a certain extent limited by the totalmass or volume of beads which has to be filled into the device in orderto achieve a therapeutically effective reduction of the concentration ofa toxin in the patient. The limitation is e.g. related to pressure dropacross the device, non-specific effects on blood components (proteinsand cells) or even storage space for the device.

(2) taking into account the different arginine contents of the beads,i.e. by taking the mass of beads in grams divided by the arginine loadin mmol/gram as a measure of the total number of ligands (FIG. 12B);this approach is justified by the fact that the arginine content is amain cost factor in the manufacturing of the ligand-polymer-matrix; theresult describes the efficiency of the ligands from an economical pointof view.

The equilibrium parameters for the different Langmuir plots are shown inTable 3. TABLE 3 Equilibrium parameters taken from the fitted curvesPS-PEG- Arg8 PS-PEG-Arg4 unit analysis affinity constant 1.81 0.15 ml/EU1 saturation 253.9 200.7 EU/g concentration analysis affinity constant1.81 0.15 ml/EU 2 saturation 479.9 168.6 EU/mmol concentrationResults and Discussion

The affinity constant describes and characterizes how strongly a certainligand is able to bind a toxin, independently of the amount of ligandpresent. Surprisingly, the affinity constant of the Arg8 ligand was morethan 10-fold higher (i.e. by a factor of 12.3) than the affinityconstant of the Arg4 ligand.

The saturation concentration describes the maximum amount of toxin whichcan be bound by the respective ligand-matrix, assuming that unlimitedamounts of toxins are available. Surprisingly the saturationconcentration of the Arg8 ligand-matrix was more than 2-fold higher(i.e. by a factor of 2.8) than the saturation concentration of the Arg4ligand-matrix. Since according to the Langmuir adsorption model thesaturation concentration is completely independent of the affinityconstant, this can be interpreted in a way that the specific geometricalarrangement of the Arg 8 ligand influences the accessibility of theligand-matrix for the toxins, e.g. by influencing the morphology (e.g.crystal-like vs. fluidic) of the PEG-part of the ligand-matrix.

The following example demonstrates the importance and relevance of theimproved affinity of the Arg8 ligand-matrix endotoxin with respect tothe amount of matrix which is necessary to achieve a therapeuticallyrelevant reduction in the toxin concentration: Endotoxin concentrationsin the range of 0.1 to 1 EU/ml are found in septic patients (e.g.Nakamura et al. Renal Failure 2000; 22: 225-234). Assuming a plasmaconcentration of 0.3 EU/ml at the start of the treatment and a plasmaconcentration of 0.03 EU/ml after treatment and a plasma volume of 4000ml, this means that 1080 EU endotoxin have to be bound to theligand-matrix during treatment. The amount of matrix necessary to bindthis amount of endotoxin can be calculated from the Langmuir expressionusing the affinity constants and the saturation concentrations for therespective ligands. The concentration at the end of the treatment isthen the equilibrium concentration. Using the equilibrium data fromTable 3 this calculation shows that for the Arg8 ligand-matrix only 83 gare necessary, whereas for the Arg4 ligand-matrix 1201 g are necessaryto achieve the therapeutic goal. Since both ligand-matrixes have asimilar density (mass per volume) this means that the volume and size ofan adsorber device will be much smaller for the Arg8 ligand, whichfacilitates perfusion by plasma or blood.

1. A polymer affinity matrix comprising a) a solid support b) at leastone spacer bound to the solid support, and, coupled to each spacer, c)at least one ligand containing at least one binding unit having at leastone functional group, wherein the polymer affinity matrix has theability to selectively bind to at least one substance in a fluid.
 2. Thepolymer affinity matrix according to claim 1, wherein said at least oneligand has a defined three-dimensional structure which is complementaryas regards charge and/or hydrophobicity/hydrophilicity to thethree-dimensional structure of a binding motif of said at least onesubstance.
 3. The polymer affinity matrix according to claim 1, whereinthe at least one ligand is represented with the formula—X¹ _(n)—Y_(m)[X² _(i)-Z¹; X³ _(j)-Z²]_(1/2(m+1)),  (general Formula I),wherein n=0 or 1; m=2^(k)˜1; k=0 to 10, wherein if k=0 then X₂═X₃ andZ₁=Z₂; i=0 or 1; and j=0 or 1, or—(X¹ _(n)—Y¹[Y² _(m[X) ² _(i)-Z¹; X³ _(j)-Z²]_(1/2(m+1)))_(r)—X⁴_(p)-Z³,  (general Formula II), wherein n=0 or 1; m=2^(k)−1; k=0-10,wherein if k=0 then X₂=X₃ and Z₁=Z₂; r=1-100; i=0 or 1; j=0 or 1; andp=0 or 1; wherein Z¹, Z² and Z³ each independently of each otherrepresents the at least one binding unit and each is an organic moleculechosen from amino acids, peptides, fatty acids, carbohydrates, lectin,and nucleotides, and derivatives thereof, and combinations thereof,wherein Y, Y¹ and Y² each is independently of each other a trifunctionalbranching molecule chosen from amino, hydroxy, aldehyde, isocyanate,isotiocyanate, thiol, maleimido, and epoxy, and derivatives thereof, andcombinations thereof, and wherein X¹, X², and X³ each, is independentlyof each other, an optional bifunctional distance molecule containing twofunctional groups chosen from amino, carboxy, hydroxy, aldehyde,isocyanate, isothiocyanate, thiol, maleimido, and epoxy, and derivativesthereof, and combinations thereof; wherein optionally the ligand iscyclic.
 4. The polymer affinity matrix according to claim 1, wherein theat least one ligand comprises 1 to 100 functional groups.
 5. The polymeraffinity matrix according to claim 1, wherein the at least one bindingunit is an amino acid, at least a part of which is positively charged atabout physiological pH of blood.
 6. The polymer affinity matrixaccording to claim 5, wherein the amino acid has a pK_(A) of ≧6.0. 7.The polymer affinity matrix according to claim 6, wherein the amino acidis arginine, lysine, histidine, or cysteine.
 8. The polymer affinitymatrix according to claim 7, wherein the amount or concentration of theamino acid is 0.01 to 5 mmol/g matrix.
 9. The polymer affinity matrixaccording to claim 8, wherein the amount or concentration of the aminoacid is chosen from 0.01, 0.1, 1, 2, 3, 4 and 5 mmol/g matrix.
 10. Thepolymer affinity matrix according to claim 5, wherein the number ofamino acid molecules per ligand is chosen from 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 and
 16. 11. The polymer affinity matrix accordingto claim 5, wherein the amino acid is arginine and the concentration ofarginine is ≦3 mmol/g matrix.
 12. The polymer affinity matrix accordingto claim 1, wherein said at least one functional group is chosen fromamino group or substituted amino group, a carboxy group, a hydroxygroup, a thiol group, a guanidino group, and combinations thereof. 13.The polymer affinity matrix according to claim 1, wherein the at leastone ligand has a tree- or comb-like structure chosen from:


14. The polymer affinity matrix according to claim 1, wherein positivecharges of at least two of the at least one functional groups areseparated from each other by a distance defined by the distance betweenindividually negatively charged groups in the binding motif of the atleast one substance.
 15. The polymer affinity matrix according to claim1, wherein the at least one spacer is substantially hydrophobic orhydrophilic and has the function of an anchoring part for the at leastone ligand.
 16. The polymer affinity matrix according to claim 15,wherein the at least one spacer is chosen from poly- or oligoethyleneglycols of the formula H—(OCH₂CH₂)_(n)—OH, wherein n represents 2 to250, polyvinylalcohols, polyvinylamines, polyolycidoles,polyethyleneimines, and polypropyleneoxides, and derivatives thereof.17. The polymer affinity matrix according to claim 16, wherein the atleast one spacer is chosen from a polyethylene glycol (PEG) in a linearand/or branched configuration and having an average molecular weight of400 to 10,000 Daltons, and derivatives thereof.
 18. The polymer affinitymatrix according to claim 1, wherein the solid support is made of amaterial chosen from polystyrene, polyvinyl alcohols,polyhydroxystyrenes, polymers produced from chloromethylatedpolystyrenes or polyacrylates, polymethacrylates functionalised withhydroxy groups, hydroxyalkyl-polystyrenes, hydroxyaryl-polystyrenes,hydroxyalkyl-aryl-polystyrenes, polyhydroxyalkylated polystyrenes,polyhydroxyarylated polystyrenes, isocyanatoalkyl-polystyrenes,isocyanatoaryl-polystyrenes, carboxyalkyl-polystyrenes,carboxyaryl-polystyrenes, aminoalkyl-polystyrenes,aminoaryl-polystyrenes, polymethacrylates, cross-linkedpolyethyleneglycols, cellulose, silica, carbohydrates, latex,cyclo-olefine copolymers, and glass and combinations thereof.
 19. Thepolymer affinity matrix according to claim 18, wherein the solid supporthas the form of a bead, gel, membrane, particle, net, woven or non-wovenfabric, fibre mat, tube, film, foil or combinations thereof orcross-linked interpenetrating networks.
 20. The polymer matrix accordingto claim 1, wherein said polymer matrix is biocompatible and has aswelling capacity enough to allow perfusion of whole blood.
 21. Thepolymer matrix according to claim 20, wherein the swelling capacity isabout 1.5 to 20 fold from a dry state to the hydrated form.
 22. Thepolymer affinity matrix according to claim 1, wherein said polymermatrix provides a three-dimensional complementary structure for bindingthe at least one substance chosen from bacteria or virus derivedconstituents; endotoxins; exotoxins; bacterial DNA and fragmentsthereof; oligonucleotides; cells; blood cells; prions; parasites; fungi;drugs after overdosing; pathogenic food additives; products from acuteor chronic metabolic disturbances resulting from diabetes mellitus,liver disease, uraemia, kidney diseases or inflammation; heparin;bacteria and viruses; pathogen-loaded blood cells; or at least parts ordegradation products thereof; DNA; phosphate; cytokines; growth factors;hormones; chemokines; uremic toxins; blood clotting proteins;procoagulatory proteins; inflammatory or proinflammatory proteins;macrophage migration inhibitory factor; soluble or cell surface boundproteins; soluble adhesion molecules; glucose or degradation productsthereof; pyrogens; bacterial exotoxins; and products from Gram-positivebacteria.
 23. The polymer affinity matrix according to any one of theclaim 1, wherein said polymer matrix has a cut-off value ranging from1×10² to 1×10⁶ Daltons and binds hydrophobic and/or hydrophilicsubstances or hydrophobic and hydrophilic substances.
 24. The polymeraffinity matrix according to claim 1, wherein the fluid an aqueous ororganic solution; a body fluid; preferably blood; therapeutical fluids;fluids for life science applications; infusion fluids or dialysis fluidsin biological, diagnostic or biotechnological applications; bloodproducts obtained from healthy donors; fluids for nutrition; and fluidsfor industrial use.
 25. The polymer affinity matrix according to claim1, wherein the solid support is a cross-linked polystyrene, the at leastone spacer is a polyethylene glycol and the at least one each bindingunit is arginine.
 26. A method for removing one or more substances froma fluid and/or reducing the amount or concentration thereof comprisingcontacting the fluid with the polymer affinity matrix of claim 1 for aperiod of time sufficient to reduce the amount or concentration orremove said at least one substance.
 27. The method according to claim26, wherein the period of time ranges from 1 to 2 hours.
 28. The methodaccording to claim 26, wherein the at least one substance is anendotoxin and the fluid is blood, wherein the amount or concentration ofendotoxin after being removed or reduced is below the capacity ofactivating components in blood or prevents activation of components orprocesses in blood.
 29. A method for producing a polymer affinity matrixas defined in claim 1, comprising a) attaching the spacer to the solidsupport to obtain a first complex, and b) attaching to said firstcomplex the ligand containing said at least one binding unit with atleast one functional group; or c) attaching the spacer to the ligandcontaining said at least one binding unit with at least one functionalgroup to obtain a second complex, and d) attaching the solid support tosaid second complex; or e) attaching the spacer to the solid support toobtain a first complex, and f) solid phase synthesis of the ligand onthe spacer bound to the solid support, or g) building up or synthesizingthe spacer from monomers directly on the solid support by grafting, andh) attaching to said first complex the ligand containing said at leastone binding unit with at least one functional group, or i) building upor synthesizing the spacer from monomers directly on the solid supportby grafting, and k) solid phase synthesis of the ligand on the spacerbound to the solid support; wherein information about three-dimensionalstructure, presence of charges and hydrophobic/hydrophilic regions ofthe binding motif on the substance to bind is collected from X-raycrystallography, protein sequencing, protein modelling or hydrophobicityand hydrophilicity calculations and the ligand containing the bindingunit is made complementary as regards charge and/orhydrophilicity/hydrophobicity to the binding motif of said substance(s).30. The method according to claim 29 comprising the steps of, for a) andb), activation of the solid support, coupling of the spacer molecule onthe solid support, synthesis of the ligand containing the binding unit,and site specific coupling of the ligand to the spacer molecule, or, forc) and d), synthesis of the ligand containing the binding unit, couplingof the spacer molecule to the ligand, activation of the solid support,and site specific coupling of the spacer-ligand complex to the solidsupport, or, for e) and f), activation of the solid support, coupling ofthe spacer molecule to the activated solid support, and solid phasesynthesis of the ligand on the spacer bound to the support.
 31. Themethod according to claim 29 comprising the steps of, for a) and b),activation of the spacer, coupling of the activated spacer to the solidsupport, and coupling the ligand to said activated spacer, or, for c)and d), synthesis of the ligand, activation of the spacer, site specificcoupling of the ligand to the activated spacer molecule and coupling ofthe spacer-ligand complex to the solid support, or, for e) and f),activation of the spacer, coupling of the activated spacer to the solidsupport and solid synthesis of the ligand on the spacer bound to thesolid support.
 32. The method according to claim 26, wherein the fluidis blood or serum.
 33. The method according to claim 32 wherein themethod results in production of less activated blood or prevention ofundesired activation of components or processes in blood.
 34. The methodaccording to claim 33, wherein the method is part of an extracorporealblood purification process or is used in an implant in the body tocontact blood or any body fluid.
 35. (canceled)
 36. A kit for removingone or more substances from a fluid or decreasing the amount and/orconcentration thereof in said fluid comprising a polymer affinity matrixas defined in claim
 1. 37. The kit according to claim 36, wherein itfurther comprises sample tubes, and a device for extra- and/orintracorporeal treatment of said fluid.
 38. A method for producing apolymer affinity matrix for removal of one or more substances from afluid or decreasing the amount or concentration thereof in said fluidwherein the specific affinity of the polymer affinity matrix isdependent on any ligand applied on the polymer matrix, wherein thepolymer matrix includes a solid support and at least one spacer, whereinthe solid support is made of a material chosen from polystyrene,polyvinyl alcohols, polyhydroxystyrenes, polymers produced fromchloromethylated polystyrenes or polyacrylates, polymethacrylatesfunctionalised with hydroxy groups, hydroxyalkyl-polystyrenes,hydroxyaryl-polystyrenes, hydroxyalkyl-aryl-polystyrenes,polyhydroxyalkylated polystyrenes, polyhydroxyarylated polystyrenes,isocyanatoalkyl-polystyrenes, isocyanatoaryl-polystyrenes,carboxyalkyl-polystyrenes, carboxyaryl-polystyrenes,aminoalkyl-polystyrenes, aminoaryl-polystyrenes, polymethacrylates,cross-linked polyethyleneglycols, cellulose, silica, carbohydrates,latex, cyclo-olefine copolymers, and glass and combinations thereof,preferably a cross-linked polystyrene, and wherein the at least onespacer is chosen from the group consisting of poly- or oligoethyleneglycols of the formula H—(OCH₂CH₂)_(n)—OH, wherein n represents 2-250.39. The method according to claim 38, wherein the solid support has theform of a bead, gel, membrane, particle, net, woven or non-woven fabric,fibre mat, tube, film, foil or combinations thereof or cross-linkedinterpenetrating networks.
 40. The method according to claim 38, whereinthe at least one spacer is chosen from a polyethylene glycol (PEG) in alinear and/or branched configuration and has an average molecular weightof 400-10 000 Daltons, and derivatives thereof.
 41. The method accordingto claim 38, wherein the polymer matrix has a swelling capacity enoughto allow perfusion of plasma or whole blood.
 42. The method according toclaim 41, wherein the swelling capacity is about 1.5 to 20 fold, from adry state to the hydrated form.
 43. The method according to claim 38,wherein the polymer matrix has the form of gel type beads.
 44. Themethod according to claim 38, wherein said fluid is an aqueous ororganic solutions; a body fluid; therapeutic fluids; fluids for lifescience applications; infusion fluids or dialysis fluids in biological,diagnostic or biotechnological application; blood products obtained fromhealthy donors; fluids for nutrition; and fluids for industrial use. 45.The method according to claim 38, wherein said polymer matrix has acut-off value ranging from 1×10² to 1×10⁶ Daltons and binds hydrophobicand hydrophilic substances or hydrophobic and/or hydrophilic substances.46. The method according to claim 38, wherein the solid support is across-linked polystyrene, and the at least one spacer is a polyethyleneglycol.